Initiation and perpetuation of inflammatory bowel diseases (IBD) may result from an exaggerated mucosal immune response to the luminal microbiota in a susceptible host. We proposed that this may be caused either 1) by an abnormal microbial composition or 2) by weakening of the protective mucus layer due to excessive mucus degradation, which may lead to an easy access of luminal antigens to the host mucosa triggering inflammation. We tested whether the probiotic Enterococcus faecium NCIMB 10415 (NCIMB) is capable of reducing chronic gut inflammation by changing the existing gut microbiota composition and aimed to identify mechanisms that are involved in possible beneficial effects of the probiotic. To identify health-promoting mechanisms of the strain, we used interleukin (IL)-10 deficient mice that spontaneously develop gut inflammation and fed these mice a diet containing NCIMB (106 cells g-1) for 3, 8 and 24 weeks, respectively. Control mice were fed an identically composed diet but without the probiotic strain. No clear-cut differences between the animals were observed in pro-inflammatory cytokine gene expression and in intestinal microbiota composition after probiotic supplementation. However, we observed a low abundance of the mucin-degrading bacterium Akkermansia muciniphila in the mice that were fed NCIMB for 8 weeks. These low cell numbers were associated with significantly lower interferon gamma (IFN-γ) and IFN-γ-inducible protein (IP-10) mRNA levels as compared to the NCIMB-treated mice that were killed after 3 and 24 weeks of intervention. In conclusion, NCIMB was not capable of reducing gut inflammation in the IL-10-/- mouse model. To further identify the exact role of A. muciniphila and uncover a possible interaction between this bacterium, NCIMB and the host in relation to inflammation, we performed in vitro studies using HT-29 colon cancer cells. The HT-29 cells were treated with bacterial conditioned media obtained by growing either A. muciniphila (AM-CM) or NCIMB (NCIMB-CM) or both together (COMB-CM) in Dulbecco’s Modified Eagle Medium (DMEM) for 2 h at 37 °C followed by bacterial cell removal. HT-29 cells treated with COMB-CM displayed reduced cell viability after 18 h (p<0.01) and no viable cells were detected after 24 h of treatment, in contrast to the other groups or heated COMB-CM. Detection of activated caspase-3 in COMB-CM treated groups indicated that death of the HT-29 cells was brought about by apoptosis. It was concluded that either NCIMB or A. muciniphila produce a soluble and heat-sensitive factor during their concomitant presence that influences cell viability in an in vitro system. We currently hypothesize that this factor is a protein, which has not yet been identified. Based on the potential effect of A. muciniphila on inflammation (in vivo) and cell-viability (in vitro) in the presence of NCIMB, we investigated how the presence of A. muciniphila affects the severity of an intestinal Salmonella enterica Typhimurium (STm)-induced gut inflammation using gnotobiotic C3H mice with a background microbiota of eight bacterial species (SIHUMI, referred to as simplified human intestinal microbiota). Presence of A. muciniphila in STm-infected SIHUMI (SIHUMI-AS) mice caused significantly increased histopathology scores and elevated mRNA levels of IFN-γ, IP-10, tumor necrosis factor alpha (TNF-α), IL-12, IL-17 and IL-6 in cecal and colonic tissue. The number of mucin filled goblet cells was 2- to 3- fold lower in cecal tissue of SIHUMI-AS mice compared to SIHUMI mice associated with STm (SIHUMI-S) or A. muciniphila (SIHUMI-A) or SIHUMI mice. Reduced goblet cell numbers significantly correlated with increased IFN-γ (r2 = -0.86, ***P<0.001) in all infected mice. In addition, loss of cecal mucin sulphation was observed in SIHUMI-AS mice. Concomitant presence of A. muciniphila and STm resulted in a drastic change in microbiota composition of the SIHUMI consortium. The proportion of Bacteroides thetaiotaomicron in SIHUMI, SIHUMI-A and SIHUMI-S mice made up to 80-90% but was completely taken over by STm in SIHUMI-AS mice contributing 94% to total bacteria. These results suggest that A. muciniphila exacerbates STm-induced intestinal inflammation by its ability to disturb host mucus homeostasis. In conclusion, abnormal microbiota composition together with excessive mucus degradation contributes to severe intestinal inflammation in a susceptible host. / Die Initiation and die Manifestation von entzündlichen Darmerkrankungen (inflammatory bowel diseases - IBD) können aus einer übersteigerten mukosalen Immunreaktion auf die luminale Mikrobiota in einem empfänglichen Wirt resultieren. Wir schlagen vor, dass dies entweder durch 1) eine abnormale mikrobielle Zusammensetzung oder 2) die Abschwächung der schützenden Schleimschicht, eingeleitet durch deren fortgeschrittenen Abbau, verursacht werden kann. Diese Entwicklung ermöglicht einen erleichterten Zugang des luminalen Antigens zu der Mukosa des Wirts und somit die Auslösung der Entzündung. Wir haben getestet, ob das probiotische Bakterium Enterococcus faecium NCIMB 10415 (NCIMB) in der Lage ist, der chronischen Darmentzündung durch Veränderung der Zusammensetzung der Darmmikrobiota entgegenzuwirken und strebten an, die zugrunde liegenden Mechanismen der probiotischen Wirkungsweise zu identifizieren. Für die Aufklärung der gesundheitsfördernden Mechanismen dieses Bakterienstammes wurden Interleukin-10 defiziente Mäuse verwendet, die spontan eine Darmentzündung entwickeln. Den Mäusen wurde für 3, 8 und 24 Wochen eine NCIMB enthaltende Diät verabreicht. Nach der Fütterung waren keine eindeutigen Unterschiede zwischen den Gruppen hinsichtlich der Genexpression von pro-inflammatorischen Zytokinen und der Zusammensetzung der Darmmikrobiota zu beobachten, obwohl eine geringere Zellzahl des schleimabbauenden Bakteriums Akkermansia muciniphila in den mit NCIMB gefütterten Mäusen nach 8 Wochen festgestellt wurde. Daraus folgt, dass NCIMB nicht in der Lage ist, dem Verlauf der Darmentzündung im IL-10-/--Mausmodell entgegenzuwirken. In der nachfolgenden Studie wurde untersucht, wie die Anwesenheit von A. muciniphila den Ausprägungsgrad einer intestinalen Salmonella enterica Typhimurium (STm) induzierten Darmentzündung beeinflusst. Dafür wurden gnobiotische C3H-Mäuse mit einem mikrobiellen Hintergrund von acht Bakterienspezies (SIHUMI) verwendet. Die gleichzeitige Anwesenheit von A. muciniphila und STm verursachte eine drastische Veränderung der Mikrobiota-Zusammensetzung des SIHUMI-Konsortiums. Diese Ergebnisse zeigen, dass A. muciniphila durch seine Fähigkeit, die Homöostase/Selbstregulation der Schleimbildung zu stören, die STm-induzierte Darmentzündung verschärft. Es kann geschlußfolgert werden, dass eine abweichende Zusammensetzung der Mikrobiota in Kombination mit einem massiven Abbau des Mucus zur schweren intestinalen Entzündung im empfänglichen Wirt beiträgt.

Orientadores: Urara Kawazoe, Elizabeth Santin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T10:12:03Z (GMT). No. of bitstreams: 1 Iuspa_MariaAparecidaMelo_M.pdf: 891258 bytes, checksum: 6e794e24b82f1c94278fc775155f9813 (MD5) Previous issue date: 2014 / Resumo: Foi realizado experimento em frangos de corte machos de duas linhagens, com duração de 42 dias. Foram utilizados 800 frangos de corte machos de um dia de idade alojados em um galpão. O delineamento experimental foi realizado em blocos, num fatorial 2 x 2 (duas linhagens infectadas e não infectadas). Cada tratamento foi replicado oito vezes. Os tratamentos experimentais foram T1, aves da linhagem Cobb 500; T2, aves da linhagem Ross 308, ambos inoculados via oral, aos 14 dias de idade em dose única de 1 x 106 oocistos; T3, aves da linhagem Cobb 500; e T4, aves da linhagem Ross 308, não inoculados. Os critérios de avaliação mensurados foram peso médio das aves, consumo de ração, conversão alimentar ajustada, eficiência alimentar, ganho de peso, escore de lesão, contagem de oocistos e análise de células caliciformes. O escore de lesão das duas linhagens infectadas foi avaliado aos seis dias após a inoculação. O número de oocistos médio por grama de excretas foi obtido entre o quarto e 10º dias após a inoculação. Os resultados de desempenho não mostraram diferenças estatísticas significativas entre as duas linhagens infectadas e as linhagens não infectadas para todos os períodos avaliados, com exceção do peso médio, aos 21 dias de idade. A linhagem Cobb apresentou um peso médio superior ao da linhagem Ross (p<0,001) aos 21 dias de idade. O escore de lesão médio da linhagem Ross foi maior que o da linhagem Cobb (p=0,006). O mesmo foi verificado para o número de oocistos médio por grama de excretas, para o qual a linhagem Ross apresentou maior contagem do que a linhagem Cobb (p<0,0001). Não houve diferença significativa na contagem de células caliciformes de duodeno e jejuno entre os tratamentos. A diferença de desempenho e resistência entre as linhagens infectadas observadas através do peso médio aos 21 dias de idade (sete dias após a inoculação), escore de lesão e quantidade de oocistos por grama de excretas desapareceu conforme o crescimento dos animais até o final do experimento. As reduções relativas de peso médio foram menores na linhagem Cobb, quando comparado à linhagem Ross, e decresceram conforme os animais chegavam à idade final do experimento nas duas linhagens (42 dias). Os aumentos relativos de conversão alimentar foram maiores para a linhagem Cobb quando comparada a linhagem Ross e também decresceram conforme os animais chegavam à idade final do experimento nas duas linhagens (42 dias). A utilização de linhagens de curva de crescimento rápido e curva de crescimento lenta de acordo com o objetivo produtivo (abate precoce ¿ frangos "griller" e abate tardio ¿ frangos pesados) deve ser considerada uma vez que as respostas de desempenho e resistência ao desafio de E. acervulina destas linhagens são distintos, podendo extrair máximo benefício da resistência genética conforme a idade de abate e desafio / Abstract: Evaluation of susceptibility and resistance between two strains of male chicken inoculated with Eimeria acervulina was carried out during 42 days. A total of 800 male broiler chickens with one day of age was used and placed in a floor-pen unit within a typical broiler house. The experimental design was in a randomized block designed in a 2 x 2 factorial (two strains infected and not infected) with eight replications. The treatments were T1, Cobb 500 broiler chicken strain; T2, Ross 308 broiler chicken strain, both were orally inoculated at 14 days of age, with a single dosage of 1 x 106 oocysts; T3, Cobb 500 broiler chicken strain; and T4, Ross 308 broiler chicken, not inoculated. The following criteria were used: average weight, feed consumption, adjustment of feed conversion, feed efficiency, weight gain, lesion score, oocysts counting, caliciform cells analysis. The results of performance did not show statistical differences between infected strains and between non infected strains during all periods, with exception to the average weight of 21 days of age. The Cobb strain showed a higher average weight compared with Ross strain (p<0.001) at 21 days of age. Lesion score evaluated at 6 days after inoculation was higher for Ross strain compared with the Cobb strain (p=0.006). The same result was observed for oocysts counting. The Ross strain presented a higher number of oocysts than the Cobb strain (p<0.0001). There was no statistical difference between strains in caliciform cells of duodenum and jejunum in all treatments. The results showed performance and susceptibility differences between infected strains on the average weight at 21 days of age (seven days after inoculation), lesion score and number of oocysts per gram of excreta; this difference disappeared according the birds were growing up, until the end of trial. The relative average of the weight reduction of the Cobb strain was lower than the Ross, and the relative average weight reduction in both strains decreased according birds were growing up, until the end of trial (42 days of age).The relative feed conversion of the Cobb strain was higher than the Ross strain and the relative feed conversion in both strains decreased according birds were growing up until the end of trial (42 days of age). The use of strains of fast growth and slow growth curve according to the production goal (early-slaughter chickens "griller" and heavy chicken) should be considered once the responses of performance and resistance to E. acervulina challenge of these strains are distinct and it is possible to have the maximum benefit from genetic resistance according to slaughter age and challenge / Mestrado / Relações Antrópicas, Meio Ambiente e Parasitologia / Mestra em Biologia Animal

<p>The objective of this thesis was to investigate the effect of dietary enzymatically treated yeast (ETY) in weanling pigs and evaluate the effect of ETY in broiler chickens during a coccidia challenge. Two studies were carried out to investigate this objective.</p> <p>The first experiment examined the effect of ETY on the growth performance, nutrient digestibility, immune response, and gut health of weanling pigs. A total of 192 weanling pigs (6.0 ± 1.04 kg) were allocated to 4 corn, soybean-based diets with increasing concentrations of ETY (0, 1, 2, or 4 g/kg) for a 43-d trial. There were 8 replicate pens (4 replicate pens per sex) and 6 pigs per replicate for each dietary treatment. The experiment was set up as a randomized complete block design. Dietary ETY supplementation did not affect the growth performance of weanling pigs. The ATTD of neutral detergent fiber (NDF) linearly increased (P < 0.05) at d 28. The concentrations of serum antioxidant and antibody markers increased (P < 0.05) from d 14 to d 43; a linear increase (P < 0.05) in catalase was observed on d 14 with increasing ETY supplementation in the diets. The inclusion of ETY in the diet did not affect the mRNA abundance of anti-inflammatory markers in the ileal mucosa of pigs but increased (P < 0.01) glutathione peroxidase 4. Ileal villus height (VH) and villus height to crypt depth (VH:CD) ratio were greater (P < 0.05) in pigs fed ETY supplemented diets relative to control pigs. However, jejunal VH and the VH:CD ratio was not affected by ETY inclusion in the diet. Pigs fed diets with increasing ETY levels had higher (P < 0.05) ileal digesta butyrate concentration relative to control pigs, but not propionate or acetate. </p> <p>The second experiment examined the effect of ETY on the growth performance, nutrient digestibility, and intestinal health of broiler chickens during a coccidia challenge. From d 1 to 14 post hatching, 480 broiler chickens (49.9 ± 3.95 g) were allocated to 3 corn-soybean meal-based diets with increasing concentrations of ETY (0, 1, or 2 g/kg). There were 16 replicate cages and 10 birds per cage. The experiment was designed as a randomized complete block design with body weight (BW) used as a blocking factor. On d 14 post hatching, the birds were combined and re-randomized within each of the 0, 1, or 2 ETY g/kg experimental diets. Following this, the number of birds was reduced to 8 birds per cage with 8 replicate cages. Each of the 3 diet groups was split into a challenge or no-challenge group. This resulted in a 3 × 2 factorial arrangement of treatments with 3 experimental diets and 2 challenge states. The BW ratio before re-randomization was maintained across all treatments during the reallotment. On d 15 post hatching, the birds in the challenge group were orally gavaged with 1 mL solution containing 25,000, 25,000, and 125,000 oocysts of E. maxima, E. tenella, and E. acervulina, respectively. The birds belonging to the no-challenge group were orally gavaged with 1 mL phosphate buffered saline. Data were analyzed using the MIXED procedure of SAS, and polynomial contrasts were used to estimate the linear and quadratic effects of ETY. The coccidia challenge (CC) decreased (P < 0.01) the BW gain, feed intake, and G:F of broiler chickens from d 14 to 21. Increasing supplementation of dietary ETY improved (P < 0.05) the G:F of birds. Also, the CC reduced (P < 0.01) the apparent total tract utilization of dry matter (DM), nitrogen (N) and gross energy (GE). The CC reduced (P < 0.01) the apparent metabolizable energy (AME) and the nitrogen corrected apparent metabolizable energy (AMEn) of diets fed to broiler chickens. On d 21, dietary ETY linearly increased (P < 0.01) the apparent ileal digestibility of DM, N, and GE in broiler chickens. The CC increased (P < 0.01) the mRNA gene expression of TNFα, IL-1β, IL-10, and IL-6. There was a tendency (P < 0.1) for ETY to reduce IL-1β expression in broiler chickens on d 21. Additionally, ETY supplementation increased (P < 0.05) the gene expression of occludin in the ceca of broiler chickens but not claudin 1. Serum catalase increased (P < 0.05) with increasing supplementation of dietary ETY in broiler chickens on d 21. Dietary ETY linearly increased (P < 0.05) the ileal villus height to crypt depth ratio and ileal goblet cell count and density in broiler chickens. The ileal and excreta oocyst counts decreased (P < 0.01) with increasing supplementation of dietary ETY in coccidia-challenged broiler chickens on d 21. </p> <p>In summary, ETY inclusion in diets of weanling pigs partially reduced reactive oxygen species damage and enhanced intestinal health without negatively affecting growth performance. Hence, ETY could be favorable in attenuating some of the deleterious effects of post-weaning stress in weanling pigs. Dietary ETY also reduced oxidative damage, improved growth performance, enhanced nutrient utilization, and augmented intestinal development in broiler chickens. However, the inclusion of ETY did not mitigate the adverse effects of a coccidia challenge in broiler chickens. Therefore, further studies may be necessary to investigate the prospect of ETY as a dietary strategy for combating coccidiosis.</p> <p><br></p>

Restrições econômicas e ambientais trazem a necessidade de substituir a farinha de peixe em dietas de organismos aquáticos por matérias-primas menos dispendiosas de origem vegetal. Entretanto, tais fontes proteicas vegetais possuem fatores antinutricionais que podem ter efeitos negativos sobre o sistema digestório dos peixes, alterando a saúde e, em consequência, a produção. O objetivo deste trabalho foi avaliar a digestibilidade e alterações na histologia do epitélio intestinal de juvenis do Characiforme carnívoro dourado, Salminus brasiliensis, alimentados com dietas contendo farelo de soja (FSO), farelo de algodão (FAl) e farelo de amendoim (FAM) como principais fontes proteicas. Foram conduzidos dois experimentos: digestibilidade (Ensaio I) e histológico (Ensaio II). O Ensaio I foi conduzido em protocolo padrão utilizando dietas práticas adicionadas do marcador inerte óxido de crômio III (CR2O3) e sistema Guelph modificado para coleta de fezes e consequente cálculo dos coeficientes de digestibilidade. No Ensaio II as mesmas fonte proteicas - FSO, FAL e FAM - foram utilizadas em substituição à proteína da farinha de peixe em uma dieta controle com cinco níveis de inclusão: 0 (controle), 25,0 %; 50%; 75,0% e 100%, em um delineamento inteiramente aleatorizado (n=3). O experimento teve a duração de 40 dias, e as coleta de amostras de tecido foram feitas aos 20 e 40 dias. As amostras do intestino posterior foram analisadas quanto às características histológicas e morfológicas, além de quantificação de células caliciformes e análise histomorfométrica do tecido intestinal. Não foram registradas diferenças para os coeficientes de digestibilidade da proteína e energia no ensaio de digestibilidade. O ensaio de desempenho mostrou que a inclusão de FAM e FAL nas dietas melhora o consumo de alimento comparativamente ao FSO Em relação as variáveis histológicas, os níveis que condicionaram alterações significativas nos parâmetros morfométricos foram FSO50 e FOS75. O FSO50 promoveu uma redução das dobras intestinais aos 20 dias, porém aos 40 dias houve aumento na altura das dobras, no espessamento da lâmina-própria e aumento na densidade das células caliciformes. Considerando a microscopia de varredura, sinais acentuados de enterite foram registrados. A alteração na morfologia e histologia do epitélio intestinal foi reflexo da interação de efeitos dos fatores antinutricionais presentes em cada fonte proteica vegetal. Consequentemente, registrou-se menor ganho de peso, crescimento e diminuição da homeostase intestinal, embora os melhores consumos e CAA fossem registrados para os peixes alimentados com FAL e FP. Conclui-se que o FSO causou enterite nos juvenis de dourado e FAL e FAM podem ser utilizados na alimentação de dourado. / Economic and environment constraints have brought the need to replace fishmeal (FM) in diets of aquatic organisms by less expensive feedstuff, especially dietary protein sources. However, such protein sources generally of plant origin, e.g. soybean meal, have antinutritional factors that can negatively affect digestive system of the fish, impairing health and consequently, production. To identify possible effects of plant protein sources in the diets for carnivore fish, this study evaluated effects of various dietary plant protein sources on digestibility, performance, histology of the intestinal epithelium of the carnivore, Neotropical Characin dourado, Salminus brasiliensis, fed plant protein-based diets. A digestibility trial was carried out in standard protocol using diets with inert marker chromium oxide III (Cr2O3) and modified system Guelph for feces. A performance test evaluated the use of three plant protein sources, soybean meal (SBM), cottonseed meal (CTM) and peanut meal (PTM) as surrogate protein source to fishmeal in a practical diet in five levels inclusion: 0 (control); 25.0%; 50.0%; 75.0% and 100%, in a randomized block design (n=3). Samples of tissue of intestinal tract of fish were collected in the distal segment at 20 and 40 days of the feeding period, and analyzed for histological and morphological characteristics. No differences were recorded for apparent digestibility coefficients (ADC) of feedstuff and energy and protein of feedstuffs. Dietary CM and PM elicited better feed consumption comparatively to the other feedstuff, Fish fed diet diet SBM50 presented reduction of intestinal folds at 20 days, but at 40 days had increased folds height, lamina propria thickening, and increased goblet cell density. Considering a scanning microscopy, evident signs of enteritis were registered. The alteration in the morphology and histology of the intestinal epithelium reflected the interaction of effects of the antinutritional factors present in each vegetable protein source, resulting in lower weight gain, growth and decreased intestinal homeostasis, even though best feed consumption and ADC were registered for fish fed CTM and FM.Concluded that SBM caused enteritis and CTM and PM can be used in feeding dourado.

Les cellules à mucus intestinales sécrètent des mucines, principalement MUC2, qui forment un gel protecteur. Malgré la place importante des acides gras à chaîne longue (AGCL) dans l'alimentation et leurs liens avec des pathologies où les cellules à mucus sont altérées il n'existe pas d'étude sur leurs impacts sur ces cellules. Mes travaux ont consisté à mettre en lumière ces interactions et à étudier leurs conséquences. In vitro, nous montrons que les AGCL saturés augmentent l'expression de MUC2 et son relarguage alors que les AGCL insaturés les inhibent et que l'acide palmitique favorise la différenciation cellulaire. In vivo, chez des ratons gavés avec de l'huile de palme, la production de MUC2 est augmentée et la perméabilité paracellulaire colique diminuée. Les huiles de colza ou de tournesol ne modifient pas la production de MUC2 et la perméabilité est identique aux contrôles malgré l'augmentation de l'expression de l'occludine. Dans un modèle de syndrome de l'intestin irritable, nous montrons que le gavage avec l'huile de palme, malgré la diminution de l'occludine, restaure la perméabilité et augmente le nombre de cellules à mucus. L'huile de colza et l'huile de tournesol ne corrigent pas le défaut de perméabilité. Parallèlement, deux études chez la souris ont mis en évidence l'impact de certains régimes sur le nombre des cellules à mucus. Ce travail a permis d'identifier les AGCL comme des nutriments capables de moduler les cellules à mucus, de faire émerger une nouvelle piste thérapeutique pour restaurer les populations de cellules à mucus et la perméabilité et d'ouvrir la réflexion sur les impacts de l'augmentation des populations de cellules à mucus chez l'individu sain / Intestinal goblet cells secrete mucins, mainly MUC2, which form a protective mucus gel. Despite the important place of long chain fatty acids (LCFA) in the diet and their links with pathologies where goblet cells are altered, there is no study dealing with their impact on goblet cells. The aim of my work was to highlight these interactions and to study their consequences. In vitro, we show that saturated LCFA increase MUC2 expression and release whereas unsaturated LCFA inhibit these process and that palmitic acid promotes cellular differentiation. In vivo, rat pups receiving oral administration of palm oil show a decrease of colonic paracellular permeability and an increase of MUC2 production. On the opposite, rapeseed and sunflower oils do not change MUC2 production and, intestinal permeability is the same as controls despite the increase of occludin expression. In an animal model of irritable bowel syndrome, palm oil effects are found again. When subjected rat pups to a maternal deprivation stress a few days after birth, they develop an intestinal hyperpermeability and a goblet cell depletion. We show that oral administration of palm oil concomitantly to the stress is able to, despite a decrease of occludin expression, restore the permeability at the level of non-stressed animals and to increase goblet cell number. Rapeseed oil and sunflower oil are not able to correct the increase of intestinal permeability. In parallel, studies in mice show that some types of diets can be associated with an increase of the number of proximal and distal goblet cells. To conclude, this work (i) helped to identify LCFA as nutrients able to modulate intestinal goblet cell number and physiology, (ii) put forward a new therapeutic track, via palmitic acid use, to restore goblet cell populations and intestinal permeability in pathologies associated with these kind of alterations and finally (iii) offered new tracks of reasoning about physiological and pathophysiological impacts of the increase of intestinal goblet cell number in a healthy subject

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-24Bitstream added on 2014-06-13T20:10:21Z : No. of bitstreams: 1 barbosa_ra_me_rcla.pdf: 1782892 bytes, checksum: c33b7a80d34353572789d680df7e20ec (MD5) / O diabetes tipo I, causado pela destruição das células beta do pâncreas, promove alterações metabólicas que podem ser acompanhadas de prejuízos em órgãos e tecidos específicos. O intestino delgado é um órgão que apresenta uma série de alterações, principalmente de caráter morfológico e funcional. A atividade física regular por sua vez, pode contribuir para o controle desta situação, sendo um fator importante, já que grande parte da população mundial sofre com esta doença. Este trabalho teve como objetivo analisar os aspectos morfológicos e histoquímicos do intestino delgado e a ultra-estrutura de células caliciformes de ratos diabéticos, a fim de se observar possíveis alterações causadas pela doença. Além disso, este trabalho buscou avaliar de que maneira o exercício físico pode interferir nestas modificações e contribuir para o controle do estado diabético. Neste estudo, ratos Wistar foram distribuídos em 4 grupos: controle sedentário (CS), controle treinado (CT), diabético sedentário (DS) e diabético treinado (DT). O diabetes foi induzido por aloxana monoidratada Sigma (35 mg/kg de peso corporal). Os grupos treinados realizaram um protocolo de exercícios físicos, que consistiu em natação com cargas acopladas ao corpo. Durante o período experimental, foram realizadas avaliações de peso corpóreo e ingestão hídrica e alimentar. Após o período experimental, o sangue dos animais foi coletado para análise de glicemia. Os intestinos delgados dos animais foram retirados, pesados e o comprimento foi medido. Amostras de duodeno, jejuno e íleo foram processadas e analisadas para a microscopia de luz e para a microscopia eletrônica de transmissão. O diabetes causou um aumento geral do intestino delgado, também devido à alteração de características histomorfométricas, tais como aumento de vilosidades e da espessura da camada mucosa... / Type I diabetes, caused by pancreatic beta cells destruction, promotes metabolic changes that may be accompanied by losses in specific organs and tissues. The small intestine is an organ that shows several changes, mainly of morphological and functional aspects. Regular physical activity can contribute to control this situation being an important factor, since most of the world population suffers from this disease. This study aimed to analyze the morphological and histochemical aspects of the small intestine and goblet cells ultrastructure in diabetic rats, in order to observe possible changes caused by the disease. In addition, this study aimed to evaluate how exercise can interfere on these changes and contribute to control the diabetic state. In this study, Wistar rats were divided into 4 groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). Diabetes was induced by alloxan monohydrate Sigma (35 mg/kg body weight). Trained groups performed an exercise protocol, which consisted of swimming with loads attached to the body. During the experimental period, assessments were made on body weight, water and food intake. After the trial period, the animals' blood was collected for glucose analysis. The small intestines of the animals were removed, weighed and the length was measured. Samples of duodenum, jejunum and ileum were processed and analyzed for light microscopy and for transmission electron microscopy. Diabetes caused a general increase in the small intestine, also due to changes on histomorphometric features, such as increased villi and increased thickness of the mucosa. In addition, the disease promoted histochemical changes as demonstrated by reduced amounts of collagen fibers, reticular fibers and general proteins, and increased amounts of neutral polysaccharides and general nucleic acids. About goblet cells, diabetes increased... (Complete abstract click electronic access below)

Submitted by LETICIA VILMA DOS SANTOS VIEIRA null (leticiavieir@hotmail.com) on 2017-09-22T15:22:13Z No. of bitstreams: 1 DISSERTAÇÃO_Letícia Vilma dos Santos Vieira.pdf: 2517417 bytes, checksum: 934c0060d04d2bb79afa7cb1c1f7b10a (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-27T18:54:34Z (GMT) No. of bitstreams: 1 vieira_lvs_me_araca.pdf: 2517417 bytes, checksum: 934c0060d04d2bb79afa7cb1c1f7b10a (MD5) / Made available in DSpace on 2017-09-27T18:54:34Z (GMT). No. of bitstreams: 1 vieira_lvs_me_araca.pdf: 2517417 bytes, checksum: 934c0060d04d2bb79afa7cb1c1f7b10a (MD5) Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Na inflamação alérgica, a histamina desencadeia papel crucial na indução de sintomas, tais como vasodilatação, broncoconstrição e produção de muco. O objetivo deste estudo foi investigar os efeitos da histamina na inflamação e no remodelamento das vias aéreas, avaliando para tanto a produção de muco e colágeno em modelo murino de inflamação pulmonar. Camundongos Balb/c machos de 20-25g foram estimulados, por via intratraqueal, com histamina em diferentes concentrações (10, 50 e 100 µM) e avaliados após 6, 24 e 48 horas. A partir dos dados resultantes do ensaio dose-resposta, foi realizado tratamento farmacológico, onde animais foram separados em 5 grupos (6 camundongos por grupo): Grupo 1 (PBS), controle não estimulado; Grupo 2 (Histamina), controle estimulado; Grupo 3 (Histamina + Loratadina); Grupo 4 (Histamina + Dexametasona) e Grupo 5 (Histamina + Dexametasona e Loratadina). Analisamos a migração de leucócitos no lavado broncoalveolar (LBA) e no tecido pulmonar. As alterações estruturais no tecido pulmonar e na traqueia foram avaliadas por análise histopatológica, bem como a produção de muco e deposição de colágeno usando Alcian blue/Periodic Acid Shiff e Tricômio de Masson, respectivamente. O sobrenadante do LBA e o tecido pulmonar foram coletados para quantificar os níveis SCF e CCL3 por ELISA de captura. A expressão gênica de Muc5ac, Gob-5, Col1a2 e receptores de histamina foi avaliada por RT-PCR em tempo real. Nossos resultados demonstram que a histamina induz inflamação das vias aéreas, com presença de infiltrado leucocitário no LBA e no tecido pulmonar. Além disso, os animais estimulados com histamina demonstraram aumento significativo na expressão gênica de Muc5ac, Gob-5 e Col1a2 no tecido pulmonar, produção SCF e CCL3 no LBA e pulmão, bem como apresentaram alterações estruturais na traqueia e no tecido pulmonar, tais como infiltrado leucocitário, presença de células caliciformes, produção de muco e depósito de colágeno. O pré-tratamento com dexametasona (DEX) inibiu a migração leucocitária no LBA e tecido pulmonar, e também diminuiu a expressão gênica de Muc5ac no tecido pulmonar, diminuiu a produção de SCF e CCL3 no LBA e pulmão, bem como reduziu a produção de muco e a deposição de colágeno no pulmão. Já o pré-tratamento com loratadina (LOR), bloqueador de H1, inibiu parcialmente a migração celular no LBA, mas não no tecido pulmonar, enquanto que a associação farmacológica entre DEX e LOR diminuiu significativamente a migração leucocitária tanto no LBA como no tecido pulmonar. Com base nos dados acima, sugere-se que a histamina induz inflamação das vias aéreas, promovendo a migração de leucócitos possivelmente através da produção de SCF e CCL3, bem como provoca alterações estruturais na traqueia e no tecido pulmonar, tais como produção de muco, deposição de colágeno, e esses efeitos tem a participação dos receptores H1 e H4. / In allergic inflammation, histamine exerts a crucial role in the induction of symptoms, such as vasodilation, bronchoconstriction and mucus production. The aim of this study was to investigate the effects of histamine in inflammation and airway remodeling, evaluating the mucus production and collagen in a murine model of pulmonary inflammation. Male Balb/c mice of 20-25g were challenged by intratracheal instillation of histamine at different concentrations (10, 50 and 100 μM) and evaluated after 6, 24 and 48 hours. From the data resulting from the dose-response assay, pharmacological treatment was performed, and animals were separated into 5 groups (6 mice per group): Group 1 (PBS), non-challenged control; Group 2 (Histamine), challenged control; Group 3 (Histamine + Loratadine); Group 4 (Histamine + Dexamethasone) and Group 5 (Histamine + Dexamethasone and Loratadine). We analyzed the recruitment of leukocytes in bronchoalveolar lavage (BAL) and lung tissue. Structural changes in lung tissue and trachea were assessed by histopathological analysis, as well as mucus production and collagen deposition using Alcian blue/Periodic Acid Shiff and Masson's Trichrome, respectively. BAL supernatant and lung tissue were collected for SCF and CCL3 levels quantification by capture ELISA. The gene expression of Muc5ac, Gob-5, Col1a2, and histamine receptors was evaluated by RT-PCR real time. Our results demonstrated that histamine induces airway inflammation, with presence of leukocyte infiltrate in BAL and lung tissue. In addition, histamine-challenged animals demonstrated a significant increase in the gene expression of Muc5ac, Gob-5 and Col1a2 in lung tissue, SCF and CCL3 production in BAL and lung, as well as structural changes in the trachea and lung tissue, such as leukocyte infiltrate, presence of goblet cell, mucus production and collagen deposition around the airways. Dexamethasone pre-treatment (DEX) inhibited leukocyte recruitment in BAL and lung tissue, decreased gene expression of Muc5ac in lung tissue, decreases SCF and CCL3 production in BAL and lung, as well as mucus production and collagen deposition in the lung. Already pre-treatment with loratadine (LOR), an H1 blocker, partially inhibited cell migration in BAL, but not in lung tissue, whereas the pharmacological association between DEX and LOR significantly reduced leukocyte recruitment in both the BAL and the lung tissue. Putting the above data together, we suggest that histamine induces airway inflammation, promoting the recruitment of leukocytes possibly through the production of SCF and CCL3, as well as causes structural changes in the trachea and lung tissue, such as mucus production, collagen deposition, and these effects might be mediated by H1 and H4 receptors.

Resumo: O diabetes tipo I, causado pela destruição das células beta do pâncreas, promove alterações metabólicas que podem ser acompanhadas de prejuízos em órgãos e tecidos específicos. O intestino delgado é um órgão que apresenta uma série de alterações, principalmente de caráter morfológico e funcional. A atividade física regular por sua vez, pode contribuir para o controle desta situação, sendo um fator importante, já que grande parte da população mundial sofre com esta doença. Este trabalho teve como objetivo analisar os aspectos morfológicos e histoquímicos do intestino delgado e a ultra-estrutura de células caliciformes de ratos diabéticos, a fim de se observar possíveis alterações causadas pela doença. Além disso, este trabalho buscou avaliar de que maneira o exercício físico pode interferir nestas modificações e contribuir para o controle do estado diabético. Neste estudo, ratos Wistar foram distribuídos em 4 grupos: controle sedentário (CS), controle treinado (CT), diabético sedentário (DS) e diabético treinado (DT). O diabetes foi induzido por aloxana monoidratada Sigma (35 mg/kg de peso corporal). Os grupos treinados realizaram um protocolo de exercícios físicos, que consistiu em natação com cargas acopladas ao corpo. Durante o período experimental, foram realizadas avaliações de peso corpóreo e ingestão hídrica e alimentar. Após o período experimental, o sangue dos animais foi coletado para análise de glicemia. Os intestinos delgados dos animais foram retirados, pesados e o comprimento foi medido. Amostras de duodeno, jejuno e íleo foram processadas e analisadas para a microscopia de luz e para a microscopia eletrônica de transmissão. O diabetes causou um aumento geral do intestino delgado, também devido à alteração de características histomorfométricas, tais como aumento de vilosidades e da espessura da camada mucosa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Type I diabetes, caused by pancreatic beta cells destruction, promotes metabolic changes that may be accompanied by losses in specific organs and tissues. The small intestine is an organ that shows several changes, mainly of morphological and functional aspects. Regular physical activity can contribute to control this situation being an important factor, since most of the world population suffers from this disease. This study aimed to analyze the morphological and histochemical aspects of the small intestine and goblet cells ultrastructure in diabetic rats, in order to observe possible changes caused by the disease. In addition, this study aimed to evaluate how exercise can interfere on these changes and contribute to control the diabetic state. In this study, Wistar rats were divided into 4 groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). Diabetes was induced by alloxan monohydrate Sigma (35 mg/kg body weight). Trained groups performed an exercise protocol, which consisted of swimming with loads attached to the body. During the experimental period, assessments were made on body weight, water and food intake. After the trial period, the animals' blood was collected for glucose analysis. The small intestines of the animals were removed, weighed and the length was measured. Samples of duodenum, jejunum and ileum were processed and analyzed for light microscopy and for transmission electron microscopy. Diabetes caused a general increase in the small intestine, also due to changes on histomorphometric features, such as increased villi and increased thickness of the mucosa. In addition, the disease promoted histochemical changes as demonstrated by reduced amounts of collagen fibers, reticular fibers and general proteins, and increased amounts of neutral polysaccharides and general nucleic acids. About goblet cells, diabetes increased... (Complete abstract click electronic access below) / Orientador: Flávio Henrique Caetano / Coorientador: Ricardo José Gomes / Banca: Dimitrius Leonardo Pitol / Banca: Cláudia Helena Pellizzon / Mestre

Restrições econômicas e ambientais trazem a necessidade de substituir a farinha de peixe em dietas de organismos aquáticos por matérias-primas menos dispendiosas de origem vegetal. Entretanto, tais fontes proteicas vegetais possuem fatores antinutricionais que podem ter efeitos negativos sobre o sistema digestório dos peixes, alterando a saúde e, em consequência, a produção. O objetivo deste trabalho foi avaliar a digestibilidade e alterações na histologia do epitélio intestinal de juvenis do Characiforme carnívoro dourado, Salminus brasiliensis, alimentados com dietas contendo farelo de soja (FSO), farelo de algodão (FAl) e farelo de amendoim (FAM) como principais fontes proteicas. Foram conduzidos dois experimentos: digestibilidade (Ensaio I) e histológico (Ensaio II). O Ensaio I foi conduzido em protocolo padrão utilizando dietas práticas adicionadas do marcador inerte óxido de crômio III (CR2O3) e sistema Guelph modificado para coleta de fezes e consequente cálculo dos coeficientes de digestibilidade. No Ensaio II as mesmas fonte proteicas - FSO, FAL e FAM - foram utilizadas em substituição à proteína da farinha de peixe em uma dieta controle com cinco níveis de inclusão: 0 (controle), 25,0 %; 50%; 75,0% e 100%, em um delineamento inteiramente aleatorizado (n=3). O experimento teve a duração de 40 dias, e as coleta de amostras de tecido foram feitas aos 20 e 40 dias. As amostras do intestino posterior foram analisadas quanto às características histológicas e morfológicas, além de quantificação de células caliciformes e análise histomorfométrica do tecido intestinal. Não foram registradas diferenças para os coeficientes de digestibilidade da proteína e energia no ensaio de digestibilidade. O ensaio de desempenho mostrou que a inclusão de FAM e FAL nas dietas melhora o consumo de alimento comparativamente ao FSO Em relação as variáveis histológicas, os níveis que condicionaram alterações significativas nos parâmetros morfométricos foram FSO50 e FOS75. O FSO50 promoveu uma redução das dobras intestinais aos 20 dias, porém aos 40 dias houve aumento na altura das dobras, no espessamento da lâmina-própria e aumento na densidade das células caliciformes. Considerando a microscopia de varredura, sinais acentuados de enterite foram registrados. A alteração na morfologia e histologia do epitélio intestinal foi reflexo da interação de efeitos dos fatores antinutricionais presentes em cada fonte proteica vegetal. Consequentemente, registrou-se menor ganho de peso, crescimento e diminuição da homeostase intestinal, embora os melhores consumos e CAA fossem registrados para os peixes alimentados com FAL e FP. Conclui-se que o FSO causou enterite nos juvenis de dourado e FAL e FAM podem ser utilizados na alimentação de dourado. / Economic and environment constraints have brought the need to replace fishmeal (FM) in diets of aquatic organisms by less expensive feedstuff, especially dietary protein sources. However, such protein sources generally of plant origin, e.g. soybean meal, have antinutritional factors that can negatively affect digestive system of the fish, impairing health and consequently, production. To identify possible effects of plant protein sources in the diets for carnivore fish, this study evaluated effects of various dietary plant protein sources on digestibility, performance, histology of the intestinal epithelium of the carnivore, Neotropical Characin dourado, Salminus brasiliensis, fed plant protein-based diets. A digestibility trial was carried out in standard protocol using diets with inert marker chromium oxide III (Cr2O3) and modified system Guelph for feces. A performance test evaluated the use of three plant protein sources, soybean meal (SBM), cottonseed meal (CTM) and peanut meal (PTM) as surrogate protein source to fishmeal in a practical diet in five levels inclusion: 0 (control); 25.0%; 50.0%; 75.0% and 100%, in a randomized block design (n=3). Samples of tissue of intestinal tract of fish were collected in the distal segment at 20 and 40 days of the feeding period, and analyzed for histological and morphological characteristics. No differences were recorded for apparent digestibility coefficients (ADC) of feedstuff and energy and protein of feedstuffs. Dietary CM and PM elicited better feed consumption comparatively to the other feedstuff, Fish fed diet diet SBM50 presented reduction of intestinal folds at 20 days, but at 40 days had increased folds height, lamina propria thickening, and increased goblet cell density. Considering a scanning microscopy, evident signs of enteritis were registered. The alteration in the morphology and histology of the intestinal epithelium reflected the interaction of effects of the antinutritional factors present in each vegetable protein source, resulting in lower weight gain, growth and decreased intestinal homeostasis, even though best feed consumption and ADC were registered for fish fed CTM and FM.Concluded that SBM caused enteritis and CTM and PM can be used in feeding dourado.

<p>Parasite infections around the world are a huge economic burden and decrease the quality of life for many people. Probiotic bacteria are being investigated as a possible treatment for many enteric issues due to their beneficial effects by altering the immune system. Goblet cells are the main source of mucins in the gut, and play an important role in host defense. Alterations in goblet cells and mucin have been implicated in a number of gastrointestinal (GI) diseases and infections. The aim of this study is to develop a probiotic based strategy to modulate goblet cell function in relation to host defense in enteric infection. Utilizing a murine model of parasite infection, <em>Trichuris muris</em>,<em> </em>we examined the effect of daily administration with probiotic <em>Lactobacillus reuteri</em> in different strains of mice and investigation of goblet cell alterations, immune and inflammatory responses in gut, and host defense mechanisms.</p> <p>Treatment with<strong> </strong>live <em>L. reuteri</em> significantly enhanced worm expulsion in resistant C57BL/6 mice and this was associated with significant increase in goblet cells numbers and an increase in IL-10. This lead to investigation of the probiotic effects in IL-10 knock out (KO) and Muc2 KO mice during the infection. There was no difference of worm burden or goblet cell amounts in infected IL-10 KO mice infected treated with probiotic or medium. In infected Muc2 KO mice treated with <em>L. reuteri</em>, there was an earlier increase of goblet cells, and a corresponding decrease in worm numbers. Finally, assessment of this probiotic in susceptible ARK mice revealed no alterations in worm burden, but the treatment prevented the increase in IFN-γ and IL-1β and significantly increased goblet cell numbers.</p> <p>These data demonstrate that altering the flora with probiotic <em>L. reuteri</em> treatment can modulate intestinal goblet cell biology and immune responses in gut, and promote worm expulsion, possibly through an IL-10 mediated mechanism. The increases in goblet cell numbers may also play a role in the early expulsion of the parasite. In addition to enhancing our understanding on the beneficial effect of probiotics in host defense in enteric infection, this research provides new information on gut function in the context of goblet cells and mucins.</p> / Master of Science (MSc)